There have been known several saccharides which are composed of glucose molecules as constituents, for example, partial starch hydrolyzates, produced from starches as materials, including amyloses, amylodextrins, maltodextrins, maltooligosaccharides, and isomaltooligosaccharides. Also, these saccharides are known to have usually non-reducing and reducing groups at their molecular ends and exhibit reducibility. Usually, partial starch hydrolyzates, which have a strong reducing power on a dry solid basis, are known to have properties of a relatively low molecular weight and viscosity, a relatively strong sweetness and reactivity, easy reactivity with amino group-containing substances such as amino acids and proteins by amino carbonyl reaction that may induce browning and unpleasant smell, and easily cause deterioration. Therefore, methods for decreasing or eliminating the reducing power of reducing saccharides without altering glucose residues have been required for a long time. For example, as disclosed in “Journal of American Chemical Society, Vol. 71, 353–358 (1949)”, it was reported that methods for forming α-, β- or γ-cyclodextrins that are composed of 6, 7 or 8 glucose molecules linked together via the α-1,4 glucosidic linkage by contacting “macerans amylase” with starches. Nowadays, these cyclodextrins are produced on an industrial scale and are used in diversified fields using their inherent properties such as non-reducibility, tasteless, and inclusion abilities. As disclosed, for example, in Japanese Patent Kokai Nos. 143,876/95 and 213,283 applied for by the same applicant of the present invention, it is known a method for producing trehalose, composed of two glucose molecules linked together via the α,α-linkage, by contacting a non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme with partial starch hydrolyzates such as maltooligosaccharides. At present, trehalose has been industrially produced from starches and used in different fields by using its advantageous non-reducibility, mild- and high quality-sweetness. As described above, trehalose having a glucose polymerization degree of 2, and α-, β- and γ-cyclodextrin having a glucose polymerization degree of 6, 7 and 8, are produced on an industrial scale and used in view of their advantageous properties, however, the types of non- or low-reducing saccharides are limited, so that more diversified saccharides other than these saccharide are greatly required.
Recently, a novel cyclic tetrasaccharide constructed by glucoses has been disclosed. For example, “European Journal of Biochemistry, Vol. 226, 641–648 (1994)” shows that a cyclic tetrasaccharide which has a structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} (hereinafter, called “cyclotetrasaccharide” in the present specification, unless specified otherwise.) is formed by contacting a hydrolyzing enzyme, alternanase, with alternan linked with glucose molecules via the alternating α-1,3 and α-1,6 bonds, followed by crystallization under the co-existence of methanol. Since cyclotetrasaccharide is a sugar having a cyclic structure and has no reducing power, it is expected that the saccharide shows no amino-carbonyl reactivity, and is useful to stabilize volatile organic compounds by its inclusion ability, and to be processed without any apprehension of browning and deterioration. However, it has been difficult to obtain alternan as a material and alternanase as an enzyme for preparing cyclotetrasaccharide. In addition, it has been substantially difficult to obtain a microorganism producing the enzyme.
Under these circumstances, the present inventors made every effort to study on a novel process for industrial production of cyclotetrasaccharide. As disclosed in PTC/JP01/04276, the present inventors found microorganisms of the genera Bacillus and Arthrobacter which produce an absolutely novel and ever unknown enzyme, α-isomaltosyl-transferring enzyme for forming cyclotetrasaccharide from a saccharide with a glucose polymerization degree of 3 or higher and bearing both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end. They found and disclosed in PCT/JP01/06412 that these microorganisms also produced another novel enzyme, α-isomaltosylglucosaccharide-forming enzyme which forms a saccharide with a glucose polymerization degree of 3 or higher and bearing both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end from saccharides with a glucose polymerization degree of 2 or higher. Furthermore, the present inventors found that cyclotetrasaccharide can be obtained from starchy saccharides with a glucose polymerization degree of 3 or higher by using α-isomaltosyl-transferring enzyme and α-isomaltosylglucosaccharide-forming enzyme. However, since the productivities of α-isomaltosyl-transferring enzyme of these microorganisms were not enough, a large-scale cultivation of these microorganisms is substantially difficult for industrial scale production of cyclotetrasaccharide.
Now, it has been revealed that the entity of the enzyme is a polypeptide, and the enzymatic activity is controlled by its amino acid sequence, as well as a DNA that encodes the amino acid sequence. Therefore, if a gene which encodes the polypeptide will be isolated, and if its nucleotide sequence will be determined, it will be relatively easy to prepare the desired amount of the polypeptide by a method which comprises the steps of constructing a recombinant DNA containing a gene which encodes the polypeptide, introducing the recombinant DNA into host-cells of microorganisms, animals or plants, and culturing the obtained transformants.
Under these circumstances, required are the isolation of a gene encoding a polypeptide as the entity of α-isomaltosyl-transferring enzyme, sequencing of the nucleotide sequence, and stable preparation of the polypeptide in large scale and at a relatively low cost.